F.prausnitzii通过MAM对溃疡性结肠炎黏膜上皮细胞紧密连接保护作用的机制研究

The strategy of bacterial therapy had been a research focus in the field of ulcerative colitis (UC) treatment, and the mechanism still unknown. Our preliminary study indicated that, the number of F. prausnitzii in the intestine of patients with UC were significantly increased after fecal microbiota transplantation (FMT), and the increasing level was correlated with the remission of intestinal inflammation and decreasing expression of myosin light chain kinase (MLCK) in colon. MLCK is a key regulator of tight junction (TJ) in epithelium cells, and the activation of MCLK leads to the damage of TJ, which will exacerbate the intestinal inflammation. Microbial anti-inflammatory molecule (MAM) is a new identified protein from the products of F. prausnitzii, which was confirmed with obvious effect in anti-inflammation. We hypothesis that, MAM protects the TJ structure of colonic epithelium cells by inhibiting the activation MLCK/MLC pathway, and which might be the mechanism of F. prausnitzii for the treatment of UC. In this study, we will observe the changing of TJ structure and the regulation of MLCK/MLC pathway by over-expression of MAM in intestinal epithelium cells. Then, bacteria harbored MAM recombinant plasmid would be transplanted into intestine of mouse colitis model, and the efficacy of MAM in protecting TJ structure through MLCK/MLC pathway will be observed in the level of histologic, protein and nucleic acid. The results of the study will help to explain the mechanism of the efficacy of F. prausnitzii in inhibiting intestinal inflammation and will provide a new strategy in the clinical treatment of UC.

溃疡性结肠炎（UC）的细菌治疗策略是近年研究热点，但机制不清。申请者前期发现：接受粪菌移植（FMT）的UC患者肠道普拉梭菌（F. prausnitzii）数量增加与炎症改善及肠黏膜MLCK表达下调密切相关。MLCK是上皮细胞紧密连接（TJ）的关键调节蛋白，其表达下降有助于改善上皮细胞TJ结构，促进UC肠黏膜愈合。但普拉梭菌通过何种机制降低MLCK表达尚不清楚。微生物抗炎分子（MAM）是新近从普拉梭菌产物中分离出的蛋白，可抑制NF-κB信号通路，产生抗炎作用。NF-κB同样是激活MLCK的上游信号分子。由此推测：普拉梭菌可通过产生MAM抑制MLCK信号通路激活，进而保护肠上皮细胞TJ结构。本项目拟在细胞过表达MAM，在结肠炎小鼠肠道定植含MAM重组质粒细菌，从基因、蛋白、组织及功能不同层次探讨MAM通过MLCK/MLC通路对肠上皮细胞TJ的保护效应，为临床治疗UC提供理论依据和新思路。

溃疡性结肠炎（UC）的细菌治疗策略是近年研究热点，但机制不清。申请者前期发现，UC患者肠道肠道菌群失调，且F. prausnitzii下降，而接受粪菌移植（FMT）的UC患者肠道普拉梭菌（F. prausnitzii）数量增加与炎症改善密切相关，提出F. prausnitzii可能在抑制肠道炎症，促进肠黏膜上皮细胞修复中发挥重要作用。但F. prausnitzii通过何种机制抑制肠道炎症尚不清楚。本项目分别从结肠类器官模型、动物模型水平验证F. prausnitzii对结肠炎的治疗作用，并发现F. prausnitzii及其代谢产物可产生相似的抑炎作用，可有效改善结肠炎症所致的小鼠体重下降，减小脾脏指数，并促进脾脏M2细胞产生，抑制M1细胞。F. prausnitzii的抗炎作用主要通过抑制IL-17A，进而抑制IL-23、GM-CSF、IL-6炎症因子分泌，抑制肠道炎症，促进肠黏膜上皮细胞的修复。同时通过菌群分析发现，在给小鼠移植入F. prausnitzii细菌之后，小鼠整体菌群发生变化，尤其以产丁酸细菌丰度上升，而促炎细菌丰度下降，提示F. prausnitzii与相关细菌之间存在协同作用，可通过细菌本身或分泌相应代谢产物促进肠道菌群重建，调节肠道免疫，进而抑制肠道炎症的作用。本研究结果首先证实F. prausnitzii及其代谢产物通过抑制IL-17A信号通路抑制肠道炎症，同时通过促进肠道菌群重建，这种细菌间的协同促进或抑制作用，将为进一步揭示其作用机制提供新的研究思路。